Aβ42 and ROS dual-targeted multifunctional nanocomposite for combination therapy of Alzheimer’s disease

Amyloid-β (Aβ) readily misfolds into neurotoxic aggregates, generating high levels of reactive oxygen species (ROS), leading to progressive oxidative damage and ultimately cell death. Therefore, simultaneous inhibition of Aβ aggregation and scavenging of ROS may be a promising therapeutic strategy to alleviate Alzheimer’s disease pathology. Based on the previously developed antibody 1F12 that targets all forms of Aβ42, we developed an Aβ42 and ROS dual-targeting nanocomposite using biodegradable mesoporous silica nanoparticles as carriers to load ultra-small cerium oxide nanocrystals (bMSNs@Ce-1F12). By modifying the brain-targeted rabies virus glycoprotein 29 (RVG29-bMSNs@Ce-1F12), this intelligent nanocomposite can efficiently target brain Aβ-rich regions. Combined with peripheral and central nervous system treatments, RVG29-bMSNs@Ce-1F12 can significantly alleviate AD symptoms by inhibiting Aβ42 misfolding, accelerating Aβ42 clearance, and scavenging ROS. Furthermore, this synergistic effect of ROS scavenging and Aβ clearance exhibited by this Aβ42 and ROS dual-targeted strategy also reduced the burden of hyperphosphorylated tau, alleviated glial cell activation, and ultimately improved cognitive function in APP/PS1 mice. Our findings indicate that RVG29-bMSNs@Ce-1F12 is a promising nanodrug that can facilitate multi-target treatment of AD. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-024-02543-z.


Introduction
Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive impairment and neuronal loss [1][2][3].Abnormal accumulation of extracellular amyloid plaques in the brain is one of the main pathological features of AD.Amyloid-β (Aβ) peptide is a major component of senile plaques, among which Aβ 42 is more prone to misfolding into different aggregates ranging in sizes from dimers to large fibrils [4,5].Although the exact role of Aβ 42 aggregates in AD pathogenesis is unclear, most Aβ 42 species are neurotoxic and lead to neuronal dysfunction and death [6][7][8][9].Accumulating evidence indicates that Aβ aggregates are found in all regions of the brain in early AD Aβ 42 and ROS dual-targeted multifunctional nanocomposite for combination therapy of Alzheimer's disease [4,10].These neurotoxic aggregates generate high levels of reactive oxygen species (ROS) leading to progressive oxidative damage and ultimately causing cell death [11,12].Indeed, there is a good correlation between elevated Aβ load and elevated levels of oxidation products in the AD hippocampus and cortex, especially in Aβ-rich brain regions [13,14].In addition, excess ROS can accelerate tau phosphorylation and Aβ production, promoting the form of neurofibrillary tangles (NFTs) and plaques [15,16].Therefore, simultaneous inhibition of Aβ aggregation and scavenging excess ROS formation may be a promising therapeutic strategy to alleviate AD pathology.
Antibodies against Aβ 42 aggregates have attracted considerable interest as potential therapeutic agents and research tools.Most anti-Aβ 42 monoclonal antibodies (mabs), such as aducanumab (recognizing low molecular weight Aβ 42 oligomer and fibrils) [17], lecanemab (recognizing Aβ 42 protofibrils) [18], and gantenerumab (recognizing insoluble Aβ 42 ) [19] preferentially target parts of Aβ aggregates rather than the monomer, suggesting that they recognize conformational epitopes.Although aducanumab and lecanemab have been approved by the FDA for AD treatment [17,18], the clinical efficacy of these antibodies against the partial conformation of Aβ 42 aggregates remains limited because the high level of Aβ 42 monomers produced during pathological conditions may rapidly misfold into different aggregate forms.Therefore, passive immunotherapy targeting all forms of Aβ 42 seems to be a promising treatment strategy.Our previously developed Aβ 42 sequence-and conformation-specific antibody 1F12 can specifically bind to soluble Aβ 42 monomers, oligomers, protofibrils, and insoluble fibers and plaque [20,21].Using this antibody against all forms of Aβ 42 species may yield desirable therapeutic effects.
However, effective delivery of therapeutics to brain Aβ-rich regions remains a major challenge due to the blood-brain barrier (BBB) allows only approximately 0.1% of peripherally administered antibodies to enter the brain [22,23].Several strategies to enhance antibody uptake in brain have been developed through receptormediated transcytosis, of which rabies virus glycoprotein 29 (RVG29) is a promising brain-targeting peptide that primarily binds to nicotinic acetylcholine receptors (nAChR) and gamma-aminobutyric acid receptors (GABAAR) located on the surface of microvascular endothelial cells and neurons [24,25].Accordingly, we hypothesized that a safe biomaterial with RVG29 and anti-Aβ 42 antibodies functionalized on its surface and loaded with ROS-scavenging agents could inhibit Aβ aggregation, accelerate Aβ 42 clearance, and scavenge ROS.We believed that biodegradable mesoporous silica nanoparticles (bMSNs), with their biodegradable nature and high loading efficiency, would be the ideal model material to test this hypothesis [26,27].bMSNs are relatively safe biomaterials characterized by high porosity, tunable pore size, high specific surface area, and easy functional modification of the surface [28,29].Further, the presence of two different oxidation states of cerium (Ce 3+ and Ce 4+ ) of cerium oxide nanoparticles (CeNPs) can mimic a series of natural redox enzymes, including superoxide dismutase (SOD) 3 and catalase, to scavenge harmful ROS from the body [30,31].The enzymatic activity of CeNPs for scavenging ROS is a result of the self-regeneration cycle of Ce 3+ /Ce 4+ and the oxygen vacancies on the cerium surface [32].Thus, bMSNs can scavenge ROS from the brain by loading CeNPs to reduce ROS-induced neuronal apoptosis, tau phosphorylation, and Aβ production, thereby inhibiting their aggregation and eventually the formation of NFTs and plaques.In addition, bMSNs can rapidly be excreted from the liver into the gastrointestinal tract [33].In this study, bMSNs were loaded with CeNPs and chemically functionalized with anti-Aβ 42 antibody 1F12 and RVG29 to bind simultaneously to Aβ 42 and scavenge ROS (Fig. 1).The specific clearance of pathogenic Aβ 42 and ROS scavenging enables the reduction of Aβ and ROS levels and attenuates a cascade of downstream damages induced by the two.The creative concept and biotechnological approach presented here open an avenue to delay the progression of AD by eliminating brain-derived Aβ 42 and ROS in the peripheral and central nervous system (CNS).

Synthesis of amine-modified biodegradable mesoporous silica nanoparticles (bMSNs)
bMSNs were synthesized according to a previously reported protocol [34,35].Briefly, 24 mL of CTAC solution (25 wt%) was first heated to 60 °C for 5 min under gentle agitation.Subsequently, TEA (0.18 g) dissolved in 2 mL of deionized water was added to the CTAC solution and kept at 60 °C for another 1 h.Then 20 mL of TEOS (20 v/v%, cyclohexane) was slowly dripped and stirred gently at 60 °C for 20 h.After cooling to room temperature, the upper organic phase was removed.The pellet was collected by centrifugation at 10,000 rpm for 10 min and washed three times with ethanol.The product was then dissolved in 50 mL of NaCl-ethanol (1 w/v%) and stirred at room temperature for 24 h to remove the template.Finally, the product was collected by centrifugation and dispersed in ethanol for further use.
To synthesize amine-modified bMSNs (NH 2 -bMSNs), 100 mg of bMSNs were dissolved in 100 mL of ethanol and heated to 88 °C for 10 min.Then, 1 mL of APTES was added and the reaction was stirred at 88 °C for 48 h.The prepared NH 2 -bMSNs were collected by centrifugation and then dispersed in DMF.

Synthesis of ceria nanoparticles (CeNPs)
Cerium (III) acetate (0.43 g) and oleylamine (3.25 g) were dissolved in 15 mL of dimethylbenzene and stirred at room temperature for 24 h.The mixture was gradually heated to 90 °C under an Ar atmosphere, and then 1 mL of deionized water was quickly added and kept at 90 °C for 3 h.Then, the mixture was rapidly cooled to room temperature and the product was pelleted by adding 100 Fig. 1 Illustration of RVG29-bMSNs@Ce-1F12 for combinational therapy of AD mL of acetone followed by centrifugation.The resulting precipitate (15 mg) was dispersed in a mixture of chloroform and DMF (50/50 v/v, 30 mL), and then BMPA (0.5 g) and citric acid (0.05 g) were added to react for another 3 h at room temperature to obtain BMAP-capped CeNPs [34].

Synthesis of bMSNs@Ce
To synthesize bMSNs@Ce, NH 2 -bMSNs (10 mg) and CeNPs (5 mg) were dissolved in DMF (10 mL).After stirring at room temperature for 12 h, a precipitate was obtained and excess CeNPs were removed by washing with DMF.The synthesized bMSNs@Ce was lyophilized in DMSO for further use.

Synthesis of bMSNs@Ce modified with RVG29 and 1F12
bMSNs@Ce with RVG29 and 1F12 modification were obtained by the following steps.First, 10 mg of bMSNs@ Ce were resuspended in 300 µL of MES buffer (100 mM MES, pH 5.0) and then 100 µL of freshly prepared EDC (50 mg/mL) and NHS (50 mg/mL) were added to homogenize by ultrasound.Then, 50 µg of RVG29 and 300 µg of anti-Aβ 42 1F12 were added to react with shaking for 4 h.Afterward, RVG29-bMSNs@Ce-1F12 was collected by centrifugation and washed twice with PBS.The synthesized RVG29-bMSNs@Ce-1F12 were stored at 4 °C until use.

Superoxide dismutase mimetic activity assay
The superoxide anion scavenging activity of RVG29-bMSNs@Ce-1F12 was measured using a superoxide dismutase (SOD) assay kit-WST (Dorento, Japan) according to the manufacturer's instructions.Briefly, RVG29-bMSNs@Ce-1F12 dispersions with different Ce element concentrations (including 0, 0.0625, 0.125, 0.25, 0.5, and 1 mM) were mixed with 200 µL of WST® working solution and added to each well.Next, 20 µL of xanthine oxidase solution was added to start the reaction by incubation at 37 °C for 20 min, and then the absorbance at 450 nm was measured using an Epoch microplate spectrophotometer (Bio Tek, USA).The SOD-mimetic activity of RVG29-bMSNs@Ce-1F12 dispersions with different concentrations of the Ce can be measured by quantifying the absorbance at 450 nm, since the absorbance is proportional to the amount of superoxide anion.

Hydroxyl radical scavenging ability assay
The hydroxyl radical scavenging ability of RVG29-bMSNs@Ce-1F12 was measured using a hydroxyl free radical scavenging ability test kit (Solarbio, China) according to the manufacturer's instructions.Briefly, RVG29-bMSNs@Ce-1F12 dispersions with different Ce element concentrations (including 0, 0.0625, 0.125, 0.25, 0.5, and 1 mM) were mixed with working buffer.The hydroxyl radical scavenging activity of RVG29-bMSNs@ Ce-1F12 dispersions with different concentrations of the Ce can be measured by quantifying the absorbance at 536 nm.

Cell culture and cell proliferation assays
SH-SY5Y and BV2 cell lines were cultured in DMEM/ F12 medium (Gibico, USA) containing 10% fetal bovine serum (FBS, Vazyme Biotech Co., Ltd, Nanjing, China), and hybridoma cells (1F12) were cultured in RMPI-1640 medium (Gibico, USA) containing 20% FBS in 5% CO 2 at 37 °C.Cell viability was measured using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, USA) according to the manufacturer's instructions.SH-SY5Y or BV2 cells (1 × 10 4 /well) were seeded in 96-well plates (NEST Biotechnology Co., Ltd, Wuxi, China) for 24 h and then treated with different concentrations of RVG29-bMSNs@Ce-1F12 dispersion from 10 to 100 µg/mL for 12 h to study biocompatibility.Untreated SH-SY5Y cells or BV2 cells were used as controls.Since absorbance is proportional to cell proliferation, cell viability was measured by quantifying the value at 490 nm.

ThT assay
Aβ 42 monomers and aggregates were prepared as previously described [21].In the presence of PBS, RVG29-bMSNs@Ce, 1F12, or RVG29-bMSNs@Ce-1F12, the process of Aβ 42 aggregation was dynamically monitored by ThT, a fluorescent probe binds specifically to β sheetrich structures and enhances fluorescence intensity [36].Briefly, aggregated Aβ 42 in each treatment group was diluted to 20 µM and mixed with 50 µM of ThT.Then, the fluorescence spectrum and intensity of the samples were detected by a multi-mode microplate reader (FlexSta-tion3; Molecular Devices, USA) under 440 nm excitation and 480 nm emission.

Inhibition of Aβ oligomerization and aggregation in vitro
Aβ 42 monomers were incubated with RVG29-bMSNs@ Ce, 1F12, or RVG29-bMSNs@Ce-1F12, respectively, while PBS-treated Aβ 42 was used as a control.Samples were incubated at 37 °C for 24 h and then centrifuged at 12,000 g for 30 min to obtain soluble Aβ 42 .The extent of oligomerization of Aβ 42 was determined by sandwich enzyme-linked immunosorbent assay (ELISA) and ThT assay.The morphology of Aβ 42 aggregates in each treatment group was observed by TEM.

Dtection of ROS levels
For the detection of ROS levels in cells, SH-SY5Y and BV2 cells at a concentration of 1 × 10 5 cells/mL were seeded in 6-well plates (NEST Biotechnology Co., Ltd, Wuxi, China) and cultured at 37 °C, 5% CO 2 for 12 h.Cells were then co-incubated with 10 µM of Aβ 42 pretreated with PBS, RVG29-bMSNs@Ce, 1F12, or RVG29-bMSNs@ Ce-1F12 at 37 °C, 5% CO 2 for 1 h.After incubation, cells from each treatment group were washed with PBS and then incubated with 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Servicebio, Wuhan, China) for 30 min at 37 °C in a 5% CO 2 incubator.After washing with PBS, the ROS fluorescence signal of cells were observed using a Zeiss LSM710 confocal laser microscope.
For the detection of ROS levels in plasma and brain tissues, the plasma samples and brain tissue supernatant collected from each treatment group were mixed with TBS buffer and then incubated with DCFH-DA (50 µM) at 37 °C for 30 min.Afterwards, a multi-mode microplate reader was used to quantitatively detect the fluorescent signals of all samples with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Cellular uptake of Aβ 42
To assess the cellular uptake of Aβ 42 in each treatment group, Cy3-labeled Aβ 42 (50 µM) was incubated with PBS, RVG29-bMSNs@Ce, 1F12, or RVG29-bMSNs@ Ce-1F12 at 37 °C for 12 h.BV2 cells were then incubated with 10 µL of the above-prepared solution containing 5 µM Aβ 42 at 37 °C for 12 h.Afterwards, cells from each group were washed with PBS, and fluorescence signals were detected by confocal laser microscopy.
After incubation, the total protein of BV2 cells was extracted using RAPI buffer (Solarbio, Beijing, China) and then quantified by NanoDrop One spectrophotometer (Thermo Fisher).The levels of Aβ 42 uptake by BV2 cells was quantitatively by sandwich ELISA.

Animal
APP/PS1 transgenic mice (Stock No: 034832-JAX) and C57BL/6J mice were ordered from Jackson Laboratory and Liaoning Changsheng biotechnology Co., Ltd, respectively.All procedures related to animal research have been reviewed and approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology.

BBB permeability in vivo
bEnd.3 (6 × 10 4 cells/transwell) was cultured in 12 wells for 7 days to form a compact monolayer to mimic the BBB.On the 5th day of culture, SH-SY5Y cells were seeded into the bottom of 12 wells.To evaluate the permeability across the monolayer, Cy3-labeled 1F12, RVG29-bMSNs@Ce, or RVG29-bMSNs@Ce-1F12 dissolved in PBS was added to the culture medium and incubated apically for 4 h.Afterwards, the fluorescence of SH-SY5Y was detected by confocal laser microscopy.

Evaluation of the effect of RVG29-bMSNs@Ce-1F12 brain targeting and Aβ plaques labeling
To evaluate the performance of RVG29-bMSNs@ Ce-1F12 brain targeting and labeling Aβ plaques in the brains of APP/PS1 mice, 14-month-old APP/PS1 mice were injected intravenously with Cy3-labeled RVG29-bMSNs@Ce-1F12 at a dose of 10 mg/kg.After 4 h of post-injection, mice were anesthetized and brain fluorescence was detected by whole-body fluorescence imaging.Mice were sacrificed after imaging and brain slices were prepared and stained with thioflavin S to evaluate the effect of RVG29-bMSNs@Ce-1F12 on Aβ plaque labeling.

In vivo degradation of bMSNs@Ce
For the biodegradation test, bMSNs@Ce NPs were dispersed in lactated Ringer's solution to a final concentration of 1 mg/mL.The mixture was incubated at 37 °C for 7 days.The resulting product was centrifuged at 10,000 rpm for 10 min and resuspended in ethanol.The degradation degree of bMSNs@Ce was observed by TEM.
Plasma or tissues from APP/PS1 mice were extracted with a tissue grinder in tris-buffered saline containing complete protease inhibitor cocktail (Roche) at a ratio of 1:50 (w/v).The homogenate supernatant was centrifuged at 12,000 g for 30 min at 4 °C to obtain TBS-soluble protein.Aβ 42 and p-tau 396,404 levels in soluble proteins were quantified by our previously prepared sandwich ELISA [21,37].

Immunoprecipitation and Western blot
Brain homogenate or plasma was incubated with 40 µg/ mL of 2C6-conjugated protein A/G magnetic beads (LinkedIn Biotechnology Co., Ltd., Shanghai, China) for 30 min at room temperature according to the manufacturer's instructions.Immunoprecipitated proteins were eluted with 0.1 M glycine (pH 3.0) and immediately neutralized to pH 7.4 with neutralization buffer (1 M Tris-HCl, pH 8.5).Samples were denatured by boiling for 10 min in loading buffer (Booster Biotech, Shanghai, China), and proteins were then run in 12% SDS-PAGE.Proteins were transferred to polyvinylidene fluoride membranes at 160 mA for 1 h.Membranes were blocked with 5% skimmed milk and incubated with 2C6 (1:2000) at 37 °C for 2 h.Membranes were then washed with PBS-T, followed by 1 h incubation with HRP-conjugated goat anti-mouse IgG (H + L) antibody (1:8000).The immune signals were visualized with ECL-substrate (Yeasen Biotechnology Co., Ltd, Shanghai, China) and detected with a Tanon 5200 Muiti (Shanghai, China).

Olfactory behavior testing and nesting
Mice were screened for olfactory deficits using the odor cross-habituation test as previously described [38].Odors (n = 6; limonene, ethyl valerate, isoamyl acetate, pentanol, heptanone, and nonane) were diluted in castor oil for olfactory behavior testing.The duration of the investigation was defined as nose-oriented sniffing within 1 cm of the odor presentation port.In the nesting test, mice in each treatment group were divided into single cages and adapted to the single-cage environment for 48 h.Then, sheets of paper (5 × 5 cm 2 ) were placed in the cage to provide conditions for mice to build their nests.Nest specific qualities were based on previously described criteria [39,40].

Morris water maze
The Morris water maze test was used to evaluate the spatial memory function of APP/PS1 mice after treatment.Before training, the pool was filled with water kept at 24-26 °C and stained with white ink.During the first five days, the mice in each treatment group were placed into the water pool from different quadrants, and they swam freely to find the hidden platform.Swim speed, swim path, and latency to find the platform were recorded.During each training session, the mice were given 60 s to find the platform.If the mice did not find the platform, they were helped to find it and allowed to stay on the platform for 15 s.The training sessions were repeated 4 times per day for five days.Finally, on the sixth day, the space exploration test was conducted by removing the hidden platform and placing the mice into the pool from the opposite side of the original platform.Their behavior within 60 s was recorded by camera and analyzed by Visutrack animal behavior analysis software (Shanghai Xinsoft Information Technology Co., Ltd).

Statistical analysis
Data are presented as mean ± SD.An unpaired t-test was used for comparisons of two groups.One-way analysis

Biocompatibility of RVG29-bMSNs@Ce-1F12 in vitro and in vivo
Nanomedicines used for AD treatment should have minimal toxicity and good biocompatibility [41].To evaluate the degradation ability of bMSNs@Ce, bMSNs@Ce was incubated with lactated Ringer's solution for different durations.The results showed that bMSNs@Ce was uniformly dispersed before degradation but became shapeless after 7 days of incubation, indicating the gradual degradation of bMSNs@Ce (Fig. 2h).The results of cell viability analysis showed that RVG29-bMSNs@Ce-1F12 showed no obvious toxicity to SH-SY5Y and BV2 cells at a concentration of 100 µg/mL (Fig. 2i).Moreover, no significant change was observed in the cell morphology of SH-SY5Y and BV2 cells after exposure to RVG29-bMSNs@ Ce-1F12 (Fig. S7a).Besides, hemolysis experiments were performed to verify the biocompatibility of the material.The results showed that when the concentration of RVG29-bMSNs@Ce-1F12 was 3.2 mg/mL, its hemolysis rate was less than 3.5%, which was 16 times higher than the injection dose (Fig. 2j and Fig. S7b).According to ISO 10993-4 [42], RVG29-bMSNs@Ce-1F12 can be considered as a feasible blood-contacting material with a hemolysis rate of less than 5%.
Considering the high liver accumulation of RVG29-bMSNs@Ce-1F12 resulting from an intravenous injection, the toxicity of RVG29-bMSNs@Ce-1F12 was further investigated in vivo via a histopathological assay (Fig. S7c).Hematoxylin-eosin-stained images of the heart, liver, spleen, lung, and kidney showed a highly similar morphology between saline-treated mouse samples and RVG29-bMSNs@Ce-1F12-treated samples.The abovementioned results indicate that RVG29-bMSNs@ Ce-1F12 has low toxicity and high biocompatibility.

RVG29-bMSNs@Ce-1F12 inhibits Aβ aggregation
Subsequently, the inhibitory effect of RVG29-bMSNs@ Ce-1F12 on Aβ aggregation was evaluated.RVG29-bMSNs@Ce, 1F12, and RVG29-bMSNs@Ce-1F12 were separately mixed with Aβ 42 and incubated for 48 h at 37 °C to test their performance.Aβ 42 was mixed with PBS and treated under the same conditions as a control.The level of Aβ 42 aggregates was detected by sandwich ELISA and ThT.Sandwich ELISA and ThT results showed that the levels of Aβ 42 aggregation was highest in the PBS group, intermediate in the RVG29-bMSNs@ Ce treatment group, and lowest in the RVG29-bMSNs@ Ce-1F12 treatment group (Fig. 3d and e).ELISA and ThT data were consistent with the dot blot analysis results, indicating a good efficiency of RVG29-bMSNs@Ce-1F12 in inhibiting Aβ 42 misfolding (Fig. S9a).On analyzing the morphology of Aβ 42 in each treatment group, we found that Aβ 42 mainly existed in the form of β-sheets and typical fibrils in the PBS control group (Fig. 3f(i)).However, in the RVG29-bMSNs@Ce treatment group, Aβ 42 mainly existed in the form of protofibrils, indicating that RVG29-bMSNs@Ce had a certain inhibitory effect on Aβ 42 aggregation (Fig. 3f(ii)).In contrast, in the 1F12 (Fig. 3f(iii)) and RVG29-bMSNs@Ce-1F12 (Fig. 3f(iv)) treatment group, only small Aβ 42 aggregates were observed and the size of the aggregates in the RVG29-bMSNs@Ce-1F12 treatment group was smaller than that in the 1F12 treatment group (Fig. S9b, p < 0.0001).These results indicated  (d-f ) ELISA, ThT, and TEM assessment of Aβ 42 aggregation in the presence of PBS, RVG29-bMSNs@ Ce, 1F12, and RVG29-bMSNs@Ce-1F12. (g) Schematic illustration of the depolymerization of Aβ 42 profibrils in the presence of RVG29-bMSNs@Ce-1F12. (h-j) ThT, ELISA, and TEM assessment of the depolymerization of Aβ 42 fibrils in the presence of RVG29-bMSNs@Ce-1F12. Data are presented as mean ± SD, n = 3. One-way analysis of variance (ANOVA) was performed for multigroup comparisons, *p < 0.05, ***p < 0.001, and ****p < 0.0001 that RVG29-bMSNs@Ce-1F12 had a good inhibitory effect on Aβ 42 aggregation.

RVG29-bMSNs@Ce-1F12 promotes the depolymerization of Aβ fibrils
A dynamic equilibrium exists between the aggregation of monomers and the dissociation of fibrils [43,44].Accordingly, we next determined whether the removal of soluble Aβ 42 by RVG29-bMSNs@Ce-1F12 could accelerate the depolymerization of fibrils.Crude Aβ 42 fibrils containing monomers and oligomers were dialyzed in PBS buffer containing RVG29-bMSNs@Ce-1F12 (20 µg/mL) to observe the dynamic dissociation of fibrils (Fig. 3g).ThT and ELISA results showed that the level of Aβ 42 fibrils gradually decreased with an increase in dialysis time (Fig. 3h and i).On analyzing the morphology of Aβ 42 in different disaggregated states, we found that the morphology of pre-dialysis samples showed β-sheets and typical fibrillar structures and this structure changed into short linear β-sheets and irregular spherical structures after 24 h of dialysis (Fig. 3j).When the dialysis duration was extended to 48 h, more irregular spherical structures were observed, indicating that Aβ fibrils had disaggregated into low-molecular-weight aggregates in the presence of RVG29-bMSNs@Ce-1F12.Thus, RVG29-bMSNs@Ce-1F12 can promote the depolymerization of insoluble Aβ 42 fibrils.

RVG29-bMSNs@Ce-1F12 alleviates Aβ aggregatesinduced oxidative stress
Misfolded Aβ 42 aggregates have been considered to be a potent mitochondrial toxicant that directly leads to mitochondrial dysfunction and neuronal apoptosis in AD [45,46].While evaluating the performance of RVG29-bMSNs@Ce-1F12 in reducing Aβ 42 aggregate-induced ROS production, DCFH-DA was used as a ROS indicator to examine ROS levels in SH-SY5Y and BV2 cells treated with PBS, RVG29-bMSNs@Ce, 1F12, and RVG29-bMSNs@Ce-1F12.As shown in Fig. 4a, c, and d, a high level of ROS was observed in PBS-treated SH-SY5Y and BV2 cells, indicating Aβ 42 aggregates can significantly induce the production of ROS.However, in the RVG29-bMSNs@Ce-1F12 treatment group, the weakest ROS fluorescence signal was observed (Fig. 4a, c, and d) due to the potent ROS-scavenging ability of RVG29-bMSNs@ Ce (Fig. 2g) and the effective inhibition of Aβ 42 aggregation by 1F12 (Fig. 3d-f ).
Studies have shown that high levels of ROS can effectively induce the proliferation of microglial cells and promote their phagocytosis [47,48].As shown in Fig. 4b and  e, the PBS group with the strongest ROS fluorescence signal had the highest Aβ 42 levels.Interestingly, the RVG29-bMSNs@Ce-1F12 treatment group with the weakest ROS fluorescence signal showed the lowest Aβ 42 levels (Fig. 4b and e).Meanwhile, the morphology of BV2 cells in the PBS-treated group with high oligomer and protofibril levels changed from a short and compact state to an extended and elongated state, resulting in decreased cell viability; however, RVG29-bMSNs@Ce-1F12 treatment reduced the proportion of elongated cells and increased cell viability (Fig. S10).Taken together, these results demonstrated the excellent ability of RVG29-bMSNs@ Ce-1F12 in reducing Aβ 42 aggregate-induced ROS production and microgliosis via potent inhibition of Aβ 42 aggregation and robust ROS scavenging.

In vitro and in vivo BBB permeability studies
RVG29-mediated BBB permeability was first studied in vitro using the endothelial bEnd.3 cell model.Cy3labeled 1F12, RVG29-bMSNs@Ce, and RVG29-bMSNs@ Ce-1F12 were incubated with compact monolayers of bEnd.3 cells grown on transwell inserts, and fluorescence imaging of SH-SY5Y cultured in the bottom chamber were performed (Fig. 5a).The results showed that fluorescent signals were observed in the RVG29-bMSNs@ Ce-and RVG29-bMSNs@Ce-1F12-treated groups, but not in the 1F12-treated group, indicating that the antibody could not penetrate the dense monolayer of bEnd. 3 cells (Fig. 5b).In contrast, RVG29-modified nanoparticles showed strong fluorescence signals, and no significant difference was observed between RVG29-bMSNs@ Ce-and RVG29-bMSNs@Ce-1F12-treated groups (Fig. 5b and c).
Furthermore, in vivo BBB permeability studies found that strong fluorescent signals were observed in the brains of C57BL/6J mice after administration of RVG29-bMSNs@Ce and RVG29-bMSNs@Ce-1F12, but not after administration of 1F12, indicating that RVG29 has good BBB permeability (Fig. 6a and Fig. S11).Meanwhile, strong fluorescent signals are also observed in the brains of 14-month-old APP/PS1 mice 4 h after the intravenous injection with RVG29-bMSNs@Ce-1F12 (Fig. 5d).Confocal fluorescence images of whole brain slices showed that brain Aβ plaques were stained by RVG29-bMSNs@ Ce-1F12 and co-localized with thioflavin S, indicating that RVG29-bMSNs@Ce-1F12 can cross the BBB and specifically recognize Aβ plaque in the brain (Fig. 5d).Altogether, the results strongly suggest that the RVG29-bMSNs@Ce-1F12 has good BBB permeability.

Evaluation of the metabolism of RVG29-bMSNs@Ce-1F12 and the pathway of the Aβ 42 clearance
To evaluate the metabolism of RVG29-bMSNs@Ce-1F12, Cy3-labeled RVG29-bMSNs@Ce-1F12 was intravenously administered to C57BL/6J mice.Cy3-labeled 1F12 and RVG29-bMSNs@Ce were used as controls.The biodistributions of the three fluorescent probes are shown in Fig. 6a and Fig. S11.The results showed that 1F12, bMSNs@Ce-RVG29, and RVG29-bMSNs@Ce-1F12 had similar metabolism pathways from the liver to the intestine.To further explore the fate of Aβ 42 cleared from peripheral blood, RVG29-bMSNs@Ce-1F12 was intravenously administered to 14-month-old APP/PS1 mice.RVG29-bMSNs@Ce was used as a control.After administration, the duodenum, jejunum, ileum, colon, caecum, and their contents were collected to prepare a homogenate, and Aβ 42 was then detected in these tissues by sandwich ELISA and IP-Western blot.ELISA results showed that Aβ 42 levels in intestinal tissues of APP/PS1 mice administered with RVG29-bMSNs@Ce-1F12 were significantly higher than those in the tissues of mice administered with RVG29-bMSNs@Ce, indicating that free Aβ 42 in the blood could be captured by RVG29-bMSNs@ Ce-1F12 and excreted it through intestinal metabolism (Fig. 6b).IP-Western blot image showed that Aβ 42 monomers and oligomers were detected in the intestine of RVG29-bMSNs@Ce-1F12 treatment group and the level of Aβ 42 isoforms was significantly higher than those treated with RVG29-bMSNs@Ce (Fig. 6c).Overall, these results demonstrate that RVG29-bMSNs@Ce-1F12 can capture peripheral soluble Aβ 42 species and excrete them through intestinal metabolism.

RVG29-bMSNs@Ce-1F12 reduces Aβ levels in the blood and brain in APP/PS1 mice
To evaluate the effect of RVG29-bMSNs@Ce-1F12 on the clearance of Aβ 42 from peripheral blood, RVG29-bMSNs@Ce-1F12 and RVG29-bMSNs@Ce (10 mg/ kg/week), and 1F12 (0.3 mg/kg/week, equal to RVG29-bMSNs@Ce-1F12 antibody amount at injection dose of 10 mg/kg) were separately administered intravenously to 14-month-old APP/PS1 mice for four weeks.Physiological saline was used in the sham control.Mouse plasma was collected and subjected to sandwich ELISA and IP-western blot to assess changes in Aβ 42 levels.Dynamic monitoring of changes in peripheral Aβ 42 levels after the administration showed a sharp decrease in Aβ 42 levels in the RVG29-bMSNs@Ce-1F12 and 1F12 treatment groups, but negligible differences were observed in the RVG29-bMSNs@Ce treatment and sham groups (Fig. 7a).After four weeks of continuous treatment, Aβ 42 was barely detectable in the RVG29-bMSNs@Ce-1F12 and 1F12 treatment groups, but high levels of soluble Aβ 42 were observed in the sham and RVG29-bMSNs@Ce treatment groups (Fig. S12).Furthermore, because of the lack of Aβ 42 targeting, there was no significant difference in plasma Aβ 42 levels between the sham group and the RVG29-bMSNs@Ce treatment group before and after treatment (Fig. 7b).The abovementioned results indicated that RVG29-bMSNs@Ce-1F12 and 1F12 can rapidly clear Aβ 42 from the peripheral blood.
We further evaluated the treatment effect of RVG29-bMSNs@Ce-1F12 in the brain.After treatment for four consecutive weeks, the brain tissues of APP/PS1 mice were collected and soluble Aβ 42 levels were assessed.ELISA results showed that the level of soluble Aβ 42 in the brains of mice treated with RVG29-bMSNs@ Ce-1F12 and 1F12 was significantly lower than that in the brains of mice treated with RVG29-bMSNs@Ce or saline, but the rate of Aβ 42 clearance by RVG29-bMSNs@ Ce-1F12 was significantly higher than that by 1F12 and RVG29-bMSNs@Ce (Fig. 7c).In contrast, brain soluble Aβ 42 levels showed negligible differences between the sham and RVG29-bMSNs@Ce treatment groups (Fig. 7c).IP-Western blot results were completely consistent with the sandwich ELISA results, indicating that RVG29-bMSNs@Ce-1F12 could significantly reduce the soluble Aβ 42 levels (Fig. 7d).Fluorescence images of coronal slices of APP/PS1 mouse brain showed that the Aβ plaque load in the hippocampus of RVG29-bMSNs@ Ce-1F12-treated mice was significantly lower than that in 1F12-and RVG29-bMSNs@Ce-treated mice, indicating that RVG29-bMSNs@Ce-1F12 effectively inhibited Aβ 42 aggregation and accelerated the clearance of Aβ 42 deposits in the brain (Fig. 7e and f ).

RVG29-bMSNs@Ce-1F12 eliminates excessive ROS and inhibits tau hyperphosphorylation in APP/PS1 mice
As the core pathogenesis of AD, oxidative stress is considered to be a key "bridge" connecting various pathogenesis and pathways of AD [49,50].In vitro test results indicated that RVG29-bMSNs@Ce-1F12 had strong ROS scavenging ability.Therefore, we evaluated ROS levels in peripheral blood and brain tissue for each treatment group.Figure 8a shows that the levels of ROS in the plasma of mice in the sham and 1F12 treatment groups were higher than those of mice in the RVG29-bMSNs@ Ce and RVG29-bMSNs@Ce-1F12 treatment groups.Similarly, this phenomenon was also observed in brain tissue, but the ROS elimination effect of the RVG29-bMSNs@Ce-1F12 treatment group was more significant than that of the RVG29-bMSNs@Ce and 1F12 groups (Fig. 8b).This may be due to the coordinated inhibition of Aβ 42 aggregation by 1F12, thereby reducing the production of ROS during the aggregation process (Fig. 4a, c,  and d) and the strong ROS scavenging ability of bMSNs@ Ce (Fig. 2g).Mounting evidence supported that the Aβ 42 aggregates and excessive ROS can induce tau hyperphosphorylation and hyperphosphorylated tau aggregation [11,51,52].Tau phosphorylation at Ser396 and Ser404 (p-tau 396,404 ) is one of the earliest events in AD and p-tau 396,404 is the main component of NFTs [53][54][55].Inhibition of tau hyperphosphorylation and hyperphosphorylated tau aggregation is an important aspect of AD treatment.Dot blot and ELISA using specific anti-p-tau 396,404 antibodies showed high levels of soluble p-tau 396,404 in the sham group [37,56], whereas treatment with RVG29-bMSNs@Ce and 1F12 resulted in decreased p-tau 396,404 levels (Fig. 8c and Fig. S13).Notably, the lowest levels of p-tau 396,404 were observed in the RVG29-bMSNs@ Ce-1F12 group (Fig. 8c and Fig. S13).In addition, fluorescence images of insoluble p-tau 396,404 in the brains of APP/PS1 mice revealed extensive p-tau 396,404 aggregates in the hippocampus of mice in the sham group (Fig. 8d  and g).In contrast, the fluorescent signal of p-tau 396,404 aggregates were significantly reduced in the brains of APP/PS1 mice treated with RVG29-bMSNs@Ce or 1F12.However, compared with RVG29-bMSNs@Ce and 1F12 treatment groups, RVG29-bMSNs@Ce-1F12 treatment group showed lower levels of p-tau 396,404 aggregates (Fig. 8d and g).These results suggested that both intravenous administration of 1F12 and RVG29-bMSNs@Ce can reduce the levels of Aβ 42 and p-tau 396,404 in the brains of APP/PS1 mice via different mechanisms.1F12 reduces brain Aβ load mainly by inhibiting Aβ 42 misfolding and attenuating Aβ-induced tau phosphorylation [57,58].In contrast, RVG29-bMSNs@Ce can eliminate excessive ROS, thereby reducing oxidative stress damage and inhibiting hyperphosphorylated tau or Aβ aggregation.Thus, RVG29-bMSNs@Ce-1F12 treatment enhanced the therapeutic effect by combining the advantages of both 1F12 and RVG29-bMSNs@Ce.

RVG29-bMSNs@Ce-1F12 attenuates microgliosis in APP/PS1 mice
Aβ and ROS in AD brains are neurotoxic and directly activate microglia, leading to neuroinflammation [4,48,59].The ability of RVG29-bMSNs@Ce-1F12 to attenuate microgliosis in vivo was evaluated.In the sham group, immunofluorescence staining of brain sections with ionized calcium-binding adaptor molecule-1 (Iba 1, a biomarker of microglia) showed a strong signal for Iba 1-positive cells, indicating that the microglia in the 14-month-old APP/PS1 mice had been activated (Fig. 8e).Compared with the sham group, the RVG29-bMSNs@Ce-1F12, 1F12, and RVG29-bMSNs@Ce treatment groups showed significantly reduced Iba 1-positive signal in the mouse brain tissues; the number and area of Iba 1-positive cells in the RVG29-bMSNs@Ce-1F12 treatment group were the lowest, indicating that RVG29-bMSNs@Ce-1F12 effectively attenuates microgliosis in vivo (Fig. 8h and i).

RVG29-bMSNs@Ce-1F12 ameliorates olfactory and cognitive dysfunction in APP/PS1 mice
Figure 8f shows the neuronal loss in dentate gyrus region, especially in Aβ plaque enriched region, but the loss of neurons was significantly improved in the RVG29-bMSNs@Ce-1F12 treatment group with low levels of Aβ plaques and ROS.It is well-known that along with the massive loss of neurons, olfactory dysfunction, memory loss, and cognitive dysfunction are typical clinical manifestations of AD [60,61].To evaluate the therapeutic effect of RVG29-bMSNs@Ce-1F12 for improving olfactory and cognitive dysfunction in 14-month-old APP/PS1 mice, a series of tests were conducted including olfactory behavior analysis, Morris water maze testing, and nestbuilding assessments (Fig. 9a).As shown in Fig. 9b, APP/ PS1 mice in the RVG29-bMSNs@Ce-1F12 treatment group took lesser time to smell the target (∼ 4.2 s), followed by the mice in 1F12 (∼ 7.6 s) and RVG29-bMSNs@ Ce (∼ 12.2 s) treatment groups, whereas the mice in the sham group took more than 18 s to smell the target, and most of the mice in the sham group failed to smell the target, indicating that RVG29-bMSNs@Ce-1F12 improved olfactory impairments.
In the Morris water maze test, the APP/PS1 mice in the sham group spent longer searching for the platform during the hidden platform stage of the tests, showing severely impaired learning and spatial memory functions (Fig. 9c).After treatment with RVG29-bMSNs@Ce-1F12, the escape latency was significantly reduced and was better than that observed in the 1F12 and RVG29-bMSNs@ Ce treatment group mice (Fig. 9c).Furthermore, there was no significant difference in swimming speed among all groups, indicating that the locomotor behavior and vision of mice were not affected after treatment with RVG29-bMSNs@Ce or 1F12 (Fig. S14a).In the stage of spatial exploration after removing the hidden platform, the mice in the sham group stayed in the platform quadrant for a shorter time than those in the RVG29-bMSNs@Ce-1F12 and 1F12 treatment groups, and their movement paths were random and disordered, indicating that these mice had severe memory dysfunction (Fig. 9d  and e).Of note, RVG29-bMSNs@Ce-1F12-treated mice exhibited a spatially oriented swimming path.They spent the longest time in the platform quadrant (Fig. 9d  and e) and crossed the platform the greatest number of times (Fig. S14b), which indicates that RVG29-bMSNs@ Ce-1F12 significantly reduced memory impairment in APP/PS1 mice.
The nest of APP/PS1 mice in the sham group was disorganized (Fig. 9f and g), indicating cognitive deficits.In contrast, the nest quality of mice in the RVG29-bMSNs@ Ce-1F12 treatment group was the highest (superior to that of mice in the 1F12 and RVG29-bMSNs@Ce treatment groups) (Fig. 9f and g).Taken together, our findings demonstrated that RVG29-bMSNs@Ce-1F12 could improve cognitive and olfactory dysfunction in APP/PS1 mice.

Conclusion
In summary, we designed and constructed a dual-targeted multifunctional nanocomposite, RVG29-bMSNs@ Ce-1F12, for combination therapy of AD.Brain-targeting RVG29 peptide and anti-Aβ 42 antibody 1F12 were immobilized on the surface of bMSNs to capture peripheral and CNS Aβ 42 and inhibit Aβ 42 oligomerization in the brain.In addition, loading CeNPs on the surface of RVG29-bMSNs@Ce-1F12 helped effectively scavenge excess ROS, thereby alleviating oxidative stress, attenuating microgliosis, and restraining hyperphosphorylated tau and Aβ aggregation.Compared with 1F12 and RVG29-bMSNs@Ce alone, the multifunctional RVG29-bMSNs@Ce-1F12 nanocomposite achieved a synergistic therapeutic effect.The encouraging results observed with RVG29-bMSNs@Ce-1F12 treatment in APP/PS1 mice indicated that Aβ 42 and ROS dual-targeted passive immunotherapy could significantly reduce multiple core pathological burdens and ameliorate cognitive impairment.This study provides insight into the design of dualtargeted multifunctional nanoplatforms with more focus on the inhibition of Aβ 42 aggregation and scavenging of ROS for AD therapy.

Fig. 9
Fig. 9 RVG29-bMSNs@Ce-1F12 ameliorates cognitive and olfactory dysfunction in APP/PS1 mice.(a) Schedule of treatment and therapeutic evaluation.(b) Investigation times for APP/PS1 mice in different treatment groups.Escape latency from the hidden platform (c), percentage of dwell time (d), and representative path tracing (e) in the platform quadrant for each treatment group.Nest quality (f ) and representative images (g) of APP/PS1 mice in different treatment groups.Data are presented as means ± SD, n = 5. *p < 0.05, **p < 0.01, and ****p < 0.0001